Mass spectrometric analysis of ligand conjugated magnetic nanoparticles

ABSTRACT

The present invention provides methods, compositions, and systems for mass spectrometric analysis of magnetic nanoparticles displaying ligands on their surface. For example, the present invention provides methods of screening a sample for the presence of at least one analyte using ligand conjugated magnetic nanoparticles, magnetic separation, and mass spectrometric analysis. The present invention also relates to MALDI matrix compositions comprising ligand conjugated magnetic nanoparticles.

The present application claims priority to U.S. Provisional ApplicationSer. No. 60/701,379 filed Jul. 21, 2005, which is herein incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to methods, compositions, and systems formass spectrometric analysis of ligand conjugated magnetic nanoparticles.For example, the present invention relates to methods of screening asample for the presence of at least one analyte using ligand conjugatedmagnetic nanoparticles, magnetic separation, and mass spectrometricanalysis. The present invention also relates to MALDI matrixcompositions comprising ligand conjugated magnetic nanoparticles.

BACKGROUND OF THE INVENTION

The completion of human genome project has catalyzed advances inproteomics to investigate cellular function at the protein level. Inparticular, increasingly sophisticated techniques have been rapidlydeveloped for discovering disease biomarkers via large-scaledifferential profiling. The recognition that every disease induces aspecific pattern of change in proteomic microenvironments indicatesimportant clinical implications on the early detection and progressionof disease. Although plasma, urine, and saliva are readily availablesamples whose protein content reflects the environment encountered bythe blood during its journey through tissues and the circulatory system,the body fluid-derived proteomes are complex, with a wide and dynamicrange in protein abundance that imposes extreme analytical difficultiesfor medical studies or clinical diagnoses. With the advent of a growingnumber of candidate protein biomarkers for disease diagnosis, thedevelopment of sensitive techniques with great potential to monitordisease onset is urgently needed for the next phase of targetedproteomics.

The detection and diagnosis of disease in the clinical setting primarilydepends on immunoassays based on antibody-antigen interactions. The mostwidely used of all the methods, enzyme-linked immunosorbent assay(ELISA), offers both specificity and sensitivity. Alternatively, proteinchip-based approaches are increasingly used in clinical diagnosis,because the array format can be easily adapted to miniaturization,multiplexing and high-throughput. However, these traditionalimmunological methods are inconvenient and time-consuming becauseenzymes or fluorescent reagents have to be labeled. Fluorescencemeasurements also may have high background, leading to false positives,and produce photobleaching, leading to false negatives (see, e.g.,Graham et al., Trends Biotechnol., 2004, 22:455-462, herein incorporatedby reference).

Recent developments in mass spectrometry have greatly expanded thepossibility of characterizing unknown proteins in proteomic research.Mass spectrometry is especially suitable for the direct detection ofproteins, which enhances specificity without the use of fluorescent orradioactive labels. This approach offers greater flexibility in theselection of bioactive probes. Among these developments, matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has become one of the primary techniques for protein identificationdue to its high sensitivity, tolerance to impurities, and high speed.Despite these advantages, the simultaneous characterization of hundredsto thousands of proteins in complex media still remains a challenge dueto the suppression effect (see Wulflkuhle et al., Nat. Rev. Cancer,2003, 3:267-275, herein incorporated by reference). Recently,surface-enhanced laser desorption/ionization (SELDI), has evolvedrapidly as a new frontier for biomarker discovery and clinical diagnosesbased on proteomic pattern analysis (see, Petricoin et al., ProteomeRes. 2004, 3:209-217, herein incorporated by reference). Despite itsadvantages of high sensitivity and high throughput, the patternrecognition platform unfortunately suffers from laboratory-to-laboratoryvariance due to differences in sample handling and analysis software(see, Diamandis et al., Mol. Cell. Proteomics, 2004, 3:367-378, hereinincorporated by reference).

As an alternative to the above approaches, MALDI MS can be combined witha biologically active probe to rapidly and specifically target proteinsof interest. This targeted approach can accelerate research forclass-specific proteins or biomarkers (Bundy et al., 2001, 73:751-757;Min et al., Nat. Biotechnol., 2004, 22:717-723; Warren et al., Anal.Chem., 2004, 76:4082-4092; and Zhang et al., Angew. Chem. Int. Ed.,2005, 44:615-617; all of which are herein incorporated by reference).Several analytical affinity capture techniques have been developed inthe field of biological mass spectrometry. The research group ofHutchens et al. was one of the first to demonstrate MS-based affinitycapture by immobilization of “bait” DNA on agarose beads for directMALDI MS analysis of targeted proteins from complex biofluids (Hutchenet al., Mass Spectrom 1993, 7:576-580, herein incorporated byreference). The concept was further tailored by Nelson and coworkers todevelop a mass spectrometric immunoassay (MSIA) (Nelson et al., Anal.Chem. 1995, 67:1153-1158, herein incorporated by reference). They usedaffinity pipette tips to selectively retrieve proteins from biologicalsolutions, demonstrating high-throughput quantitative protein analysisas well as screening of heterogeneous glycan structures in plasmaproteins (Nedelkow et al., Anal. Chem., 2004, 76:1733-1737; and Kiernanet al., Proteomics, 2004, 4:1825-1829, both of which are hereinincorporated by reference). Variations of the biologically active probesfor affinity mass spectrometry include the assay of directdesorption/ionization on silicon (DIOS) (Wei et al., Nature, 1999,399:243-246, and Zou et al., Angew. Chem. Int. Ed. 2002, 41:646-648,both of which are herein incorporated by reference) and self-assembledmonolayers (SAMs) (Brockman et al., Anal. Chem. 1995, 67, 4581-4585; andSu et al., Angew. Chem. Int. Ed. 2002, 41:4715-4718, both of which areherein incorporated by reference). Despite the rapid evolution ofefficient chip-based or microbead-based assays for biomedical research,protein chip technologies face two main technical challenges. First, thephysical and chemical properties of the chip surface may denature/alterthe native three-dimensional structure of proteins, raising thepossibility of disrupted bait-target protein interactions. Secondly, therequirement of specialized immobilization chemistry for surfaceengineering and/or specialized instruments limit the general applicationof these protein assay technologies in the general scientific community.Therefore, what is needed is a detection assay and associatedcompositions that avoid these problems.

SUMMARY OF THE INVENTION

The present invention provides methods, compositions, and systems formass spectrometric analysis of ligand conjugated magnetic nanoparticles.For example, the present invention provides methods of screening asample for the presence of at least one analyte using ligand conjugatedmagnetic nanoparticles, magnetic separation, and mass spectrometricanalysis. The present invention also relates to MALDI matrixcompositions comprising ligand conjugated magnetic nanoparticles.

In some embodiments, the present invention provides methods of screeninga sample comprising; a) providing; i) a first population of magneticnanoparticles, wherein the magnetic nanoparticles display (e.g., areconjugated to) a plurality of ligand molecules, and ii) a sample (e.g.biological sample) suspected of containing at least one type of targetanalyte; and b) mixing the first population of magnetic nanoparticleswith the sample; c) separating (e.g., magnetically separating) at leasta portion of the first population of magnetic nanoparticles from thesample thereby generating a second population of magnetic nanoparticles,and d) subjecting at least a portion of the second population ofmagnetic nanoparticles to mass spectrometric analysis under conditionssuch that the presence or absence of the at least one analyte in thesample is detected.

In certain embodiments, the methods further comprise a step after stepc), but before step d), of washing and/or eluting the second populationof magnetic nanoparticles. In other embodiments, at least a portion ofthe second population is mixed with matrix material, wherein the matrixmaterial is configured for use in matrix assisted laserdesorption-ionization (MALDI) mass spectrometry (e.g. MALDI-TOF orsimilar techniques). In particular embodiments, the mass spectrometricanalysis comprises matrix assisted laser desorption-ionization (MALDI)mass spectrometry or similar type of mass spectrometry. In otherembodiments, the mass spectrometric analysis comprises time of flightmatrix assisted laser desorption-ionization (MALDI-TOF) massspectrometry or similar method.

In particular embodiments, the presence of the analyte is detected andthe mass spectrometric analysis determines an approximate amount of theat least one analyte in the sample (e.g., the method is quantitative orsemi-quantitative). In some embodiments, the mass spectrometric analysisis multiplexed and detects the presence of at least two types ofanalytes (e.g. 2, 5, 10, 50, 100, or 1000 different types of analytesare detected). In some embodiments, the first population of magneticnanoparticles comprises a first sub-population conjugated to ligandsspecific for one type of analyte and a second sub-population conjugatedto ligands specific for a second type of analyte. In other embodiments,the first population of magnetic nanoparticles comprises a first,second, third, fourth, fifth, sixth . . . one-hundredth sub-populationconjugated to a unique ligand specific for a particular analyte. Inadditional embodiments, the plurality of ligand molecules comprise atleast two different types of ligand molecules (e.g. such that eachmagnetic nanoparticle is able to bind with two types of targetanalytes). In certain embodiments, the plurality of ligand moleculescomprise at least three, four, five . . . one-hundred different types ofligand molecules (e.g. such that each magnetic nanoparticle is able tobind with three, four, five, etc. types of target analytes).

In particular embodiments, the at least one analyte is present in thesample at a concentration equal to or less than 1×10⁻⁷ M, and whereinthe presence of the at least one analyte is detected. In someembodiments, the at least one analyte is present in the sample at aconcentration equal to or less than 1×10⁻⁸ M or 1×10⁻⁹ M (or between1×10⁻⁶ M and 1×10⁻¹⁰ M or between 1×10⁻⁷ M and 1×10⁻⁹ M) and thepresence of the at least one analyte is detected. In additionalembodiments, the total volume of the sample is less than 10 μl. Infurther embodiments, the total volume of the sample is between 0.5 μland 10 μl. In some embodiments, the total weight of the first populationof magnetic nanoparticles is equal to or less than 10 μg (e.g. 10, 8, 6or 4 μg). In further embodiments, the total weight of the firstpopulation of magnetic nanoparticles is between 10 μg and 5 μg. Incertain embodiments, the sample comprises blood plasma. In otherembodiments, the sample comprises fluid obtained from a subject (e.g.urine, blood, blood plasma, semen, stool, or any other type of fluidfrom a subject).

In some embodiments, the present invention provides compositionscomprising; a) a population of magnetic nanoparticles, wherein themagnetic nanoparticles display (e.g., are conjugated to) a plurality ofligand molecules; and b) matrix material, wherein the matrix material isconfigured for use in mass spectrometric analysis (e.g., in matrixassisted laser desorption-ionization (MALDI) mass spectrometry). Incertain embodiments, at least a portion of the ligands are bound toanalyte molecules.

In particular embodiments, the present invention provides systemscomprising; a) a population of magnetic nanoparticles, wherein themagnetic nanoparticles display (e.g., are conjugated to) a plurality ofligand molecules; and b) a mass spectrometric device, wherein the massspectrometric device is configured to detect the presence or absence ofat least one type of analyte bound to the ligand molecules. In certainembodiments, the mass spectrometric device is configured for matrixassisted laser desorption-ionization (MALDI) mass spectrometry. In someembodiments, the mass spectrometric device is configured for time offlight matrix assisted laser desorption-ionization (MALDI-TOF) massspectrometry.

In certain embodiments, the plurality of ligand molecules comprisesantibodies or antibody fragments. In further embodiments, the pluralityof ligand molecules comprise proteins (e.g. receptors, antibodies,antibody fragments, etc), carbohydrate molecules, or nucleic acids (e.g.nucleic acids that are known to bind proteins, or nucleic acids found bymethods such as the SELEX method). In some embodiments, the magneticnanoparticles comprise blocking molecules (e.g. BSA, polyethyleneglycol, methoxy polyethylene glycol, etc.). In other embodiments, theplurality of ligand molecules are conjugated to the magneticnanoparticles via a linker molecule. (e.g. as shown in FIG. 1 and FIG.9).

In particular embodiments, the matrix material has a pH of 2.0 or less(e.g. a pH of 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.3, 1.1, or 1.0). Infurther embodiments, the matrix material is selected from the groupconsisting of: nicotinic acid; glycerol; sinapinic acid; ferulic acid;caffeic acid; succinic acid; 2,5-dihydroxy benzoic acid;α-cyano-4-hydroxy cinnamic acid; 3-hydroxypicolinic acid,2-(4-hydroxyphenylazo)-benzoic acid; 2,4,6-trihydroxy-acetophenone;3-amino-4-hydroxy benzoic acid; 5-methoxysalicylic acid; 1-hydroxyisoquinoline; 2,6-dihydroxyacetophenone,4-hydroxy-3-methoxyphenylpyruvic acid; indole-3-pyruvic acid; harmaline;3-aminoquinilone; and compositions similar to any of these compounds.

In certain embodiments, the magnetic nanoparticles comprise a metalliccore particle. In other embodiments, the metallic core particle has adiameter of 1 to 150 nanometers. In some embodiments, the metallic coreparticle has a diameter of 5 to 15 nanometers. In further embodiments,the metallic core particle has a diameter of 0.1 to 500 nanometers(e.g., 0.1 nm, 0.5 nm, 1.0 nm, 2 nm, 2.5 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 18 nm, 20 nm,25 nm, 50 nm, 75 nm, 100 nm, 150 mm, 200 nm, 250 nm, 300 nm, 400 nm and500 nm). In particular embodiments, the metallic core particle comprisesa silicon coating. In some embodiments, the metallic core particlecomprise iron (e.g., Fe₃O₄). In certain embodiments, the metallic coreparticle comprises a material selected from iron, nickel, cobalt, andalloy of these metals. In some embodiments, the magnetic nanoparticlescomprise a ceramic core particle, wherein the core ceramic core particlehas magnetic properties. In preferred embodiments, the magneticnanoparticles comprise a core particle that exhibit superparamagneticproperties.

In some embodiments, the present invention provides kits comprising i) apopulation of magnetic nanoparticles, wherein the magnetic nanoparticlesare conjugated to a plurality of ligand molecules, and ii) instructionsfor using the magnetic nanoparticles with mass spectrometric devices(e.g. instructions for therapeutic, diagnostics, or basic research useof the magnetic nanoparticles with mass spectrometric devices).

One feature of the screening methods of the present invention is theadvantage of on-probe identification of unknown target proteins (orother molecules) by mass spectrometry or identifying binding epitopes ontarget analytes. In some embodiments, the present invention providesmethods of screening a sample for target ligand binding moleculescomprising; a) providing; i) a first population of magneticnanoparticles, wherein the magnetic nanoparticles display (e.g., areconjugated to) a plurality of ligand molecules, and ii) a sample (e.g.biological sample) comprising candidate ligand binding molecules; and b)mixing the first population of magnetic nanoparticles with the sampleunder conditions such that at least one type of target ligand bindingmolecule binds to the ligand molecules; c) separating (e.g.,magnetically separating) at least a portion of the first population ofmagnetic nanoparticles from the sample thereby generating a secondpopulation of magnetic nanoparticles, and d) subjecting at least aportion of the second population of magnetic nanoparticles to massspectrometric analysis under conditions such that data regarding the atleast one target ligand binding molecule is generated. In certainembodiments, the data comprises information on the mass of the at leastone target ligand binding molecule. In other embodiments, the datacomprises information on the mass of one or more fragments of the atleast one target ligand binding molecule. In further embodiments, thetarget ligand binding molecule comprises a protein. In certainembodiments, the present invention provides methods of characterizingligand molecule binding epitopes in a target molecule comprising; a)providing; i) a first population of magnetic nanoparticles, wherein themagnetic nanoparticles display a plurality of ligand molecules, and ii)a sample comprising candidate ligand binding molecules; and b) mixingthe first population of magnetic nanoparticles with the sample underconditions such that at least one type of target ligand binding moleculebinds to the ligand molecules; c) exposing said first population ofmagnetic nanoparticles to a digestion agent; d) magnetically separatingat least a portion of the first population of magnetic nanoparticlesfrom the sample thereby generating a second population of magneticnanoparticles, and e) subjecting at least a portion of the secondpopulation of magnetic nanoparticles to mass spectrometric analysisunder conditions such that data regarding at least one ligand bindingmolecule epitope is generated.

The mass spectrometric data generated by the methods of the presentinvention can be used to determine the identity of the target ligandbinding molecule, using, for example, the MS-Fit database search enginewith 100% sequence coverage. Additional details on MS-Fit, as well asthe software used for MS-FIT, can be found on the internet at MS fit at“http://” followed by “prospector.ucsf.edu.” The data generated by thepresent invention can be used, for example, with MS-FIT or similarprograms to identify target proteins or binding epitopes.

DESCRIPTION OF THE FIGURES

FIG. 1A shows one embodiment of the synthesis of an antibody conjugatedmagnetic nanoparticle, and FIG. 1B shows a nanoparticlesize-distribution histogram of the particles having an average diameterof 10 nm.

FIG. 2 shows a MALDI mass spectra of a protein mixture before (A) andafter using (B) anti-CRP- or (C) anti-SAP-conjugated mNPs to extract aspecific protein. The protein solution (60 μL) was composed of 0.5 μMmyoglobin (Myo), 0.1 μM C-reactive protein (CRP), 0.67 μM serum amyloidP component (SAP), and 2.1 μM enolase (Eno). The arrow in the massspectrum of (A) indicates the theoretical m/z of CRP. The inset of (C)shows detailed protein expression profiles of wild type, monosialo-, andasialo-SAP.

FIG. 3 shows the effect of incubation time on antibody-antigenrecognition using antibody-conjugated mNPs. To investigate the timecourse of antibody-antigen recognition on mNPs, 1 μL of supernatant wassampled from a 60-μL reaction after different incubation times. The mNPswere conjugated with: anti-SAP (squares with solid line) and anti-CRP(circles with dashed line). After incubation (3 min to 1 hr), thequantities of the antigen remaining in the supernatant were detected byMALDI MS, and the peak intensities were plotted as a function ofincubation time.

FIG. 4 shows a MALDI mass spectra of serum amyloid P component (SAP)extracted from diluted solution using anti-SAP-conjugated mNPs: (A) 160nM SAP, 50 μL; (B) 40 nM, 200 μL; (C) 16 nM, 500 μL; (D) 8 nM, 1000 μL.The inset of each panel shows the mass spectrum of solution prior toextraction.

FIG. 5 shows the MALDI mass spectra of serum amyloid P component (SAP)extracted from 60 μL of protein solution of (A) 1.9 μM, (B) 80 nM, (C)15 nM, (D) 3 nM, (E) 0.6 nM, using anti-SAP-conjugated mNPs.

FIG. 6 shows a comparison of affinity extraction betweenantibody-conjugated magnetic nanoparticles and magnetic microbeads: (A)MALDI mass spectrum of 200-fold-diluted plasma; high-abundance proteinswere so dominant that the signals from SAP or CRP were buried in thespectrum. Representative mass spectra of (B) SAP and (C) CRP wereobtained after extraction using antibody-conjugated mNPs. Spectra forantibody-conjugated microbeads are shown in (D) SAP and (E) CRP.

FIG. 7 shows a MALDI mass spectra of SAP extracted from 1 μL humanplasma, either undiluted (A), diluted 50-fold (B), diluted 500-fold (C),or diluted 1000-fold (D), using anti-SAP-conjugated mNPs extraction. Theinset of each panel shows the mass spectrum of solution prior toextraction.

FIG. 8 shows results of screening of human plasma from healthyindividuals and patients with gastric cancer using thenanoparticle-based mass spectrometric immunoassay. The MALDI massspectra of CRP, in the m/z range of 20,000-25,000, are shown in panel(A). The MALDI mass spectra of SAP, in the m/z range of 24,000˜27,000,are shown in panel (B).

FIG. 9 shows one embodiment of the preparation of antibody-conjugatedmagnetic nanoparticles of the present invention and process forimmunoaffinity assay.

FIG. 10 shows a MALDI-TOF mass spectra of human plasma fromimmunoaffinity extraction with anti-SAP MNP: (A) 25-fold dilution ofhuman plasma (B) extraction by anti-SAP-MNP (w/o blocking); (C)extraction by anti-SAP MNP (BSA blocking); and (D) extraction byanti-SAP MNP (MEG blocking).

FIG. 11 shows the concentration effect of MEG blocking of mNPs.

FIG. 12 shows a MALDI-TOF MS spectrometry of (A) human serum (containingCRP 5 mg/L) extracted by anti-CRP-MNP (w/o blocking) (B) human serum(containing CRP 5 mg/L) extracted by anti-CRP-MNP (with 30 mM MEGblocking) (C) the extreme low abundant CRP (<0.9 mg/L) of healthy humanserum extracted by anti-CRP-MNP (w/o blocking) (D) the extreme lowabundant CRP (<0.9 mg/L) of healthy human serum extracted byanti-SAP-MNP (with 30 mM MEG blocking).

FIG. 13 shows a MALDI-TOF mass spectrum obtained from multiplexedimmunoassay of SAA, CRP, and SAP from human plasma sample as describedin Example 3.

DEFINITIONS

To facilitate an understanding of the invention, a number of terms aredefined below.

As used herein, the terms “subject” and “patient” refer to any animal,such as a mammal like a dog, cat, bird, livestock (e.g. pig), andpreferably a human.

The term “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture (e.g., microbiological cultures). On the other hand, it is meantto include both biological and environmental samples (e.g., blood plasmasample). A sample may include a specimen of synthetic origin.

Biological samples may be animal, including human, fluid, solid (e.g.,plasma) or tissue, as well as liquid and solid food and feed productsand ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Biological samples may be obtained from all ofthe various families of domestic animals, as well as feral or wildanimals, including, but not limited to, such animals as ungulates, bear,fish, lagamorphs, rodents, etc.

Environmental samples include environmental material such as surfacematter, soil, water and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,utensils, disposable and non-disposable items. These examples are not tobe construed as limiting the sample types applicable to the presentinvention.

As used herein, the term “magnetic nanoparticle” refers to smallparticles (e.g. nanometer range) that are magnetic and effectively serveas a solid support or solid phase for conjugation to a ligand molecules.Even though particles can be of any size, the preferred size is 0.1-500nanometers, preferably 1-150 nanometers, more preferably 5-15nanometers, and most preferably about 9.0-10.0 nanometers. The particlesmay be uniform (e.g., being about the same size) or of variable size.Particles may be any shape (e.g. spherical or rod shaped), but arepreferably made of regularly shaped material (e.g. spherical). Theparticles of the present invention are preferably composed of materialthat exhibits superparamagnetic properties (see, e.g., Spinu et al.,IEEE Transactions on Magnetics, 36(5):3032-3034, 2000, hereinincorporated by reference).

As used herein, the term “target analyte” refers to a molecule in asample to be detected or targeted by magnetic nanoparticles. Examples oftarget molecules include, but are not limited to, cell surface ligands,cells in a subject, pathogens, such as bacteria and viruses, antibodies,naturally occurring drugs, synthetic drugs, pollutants, allergens,affector molecules, growth factors, chemokines, cytokines, andlymphokines. Preferably, the target analysts are found in blood plasma(e.g. human blood plasma).

The term “antibody,” as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL.

As used herein, the term “antibody fragments” refers to a portion of anintact antibody. Examples of antibody fragments include, but are notlimited to, linear antibodies, single-chain antibody molecules, Fab andF(ab′)₂ fragments, and multispecific antibodies formed from antibodyfragments. The antibody fragments preferably retain at least part of theheavy and/or light chain variable region.

As used herein, the phrase “matrix material” refers to material used toembed compounds to be analyzed by matrix assisted type massspectrometric analysis (e.g. MALDI-TOF). Matrix material may be, forexample, an organic acid with a strong absorption at the wavelength ofthe laser being used as part of mass spectrometric analysis.

As used herein, the terms “ligand” and “ligand molecule” refer to anymolecule that is able to bind to another molecule (e.g. a targetanalyte). Preferably, the ligand molecules of the present invention aredisplayed on the surface of a magnetic nanoparticle (e.g. covalentlyattached via a linker to a magnetic nanoparticle). Example of ligandmolecules include, but are not limited to, proteins (e.g. antibodies,antibody fragments or receptors), carbohydrates, lipids, or nucleicacids.

As used herein, the phrase “mass spectrometric analysis” refers to anymethod for identifying chemical composition of substances by use of amass spectrometer, where a mass spectometer is a device that usemagnetic fields, electric fields, or both to determine the masses ofisotopes in a sample by producing a mass spectrum.

DESCRIPTION OF THE INVENTION

The present invention provides methods, compositions, and systems formass spectrometric analysis of magnetic nanoparticles displaying ligandmolecules. For example, the present invention provides methods ofscreening a sample for the presence of at least one analyte using ligandconjugated magnetic nanoparticles, magnetic separation, and massspectrometric analysis. The present invention also relates to MALDImatrix compositions comprising ligand conjugated magnetic nanoparticles.

The ligand conjugated magnetic nanoparticles (mNPs) of the presentinvention, particularly antibody conjugated magnetic nanoparticles, arewell suited for use with mass spectrometric analysis of biologicalsamples (e.g. screening plasma samples). For example, as described inthe Examples section, the present inventors have shown that covalentconjugation of antibodies to mNPs yields a stable affinity probe formass spectrometric analysis (e.g. immunoassay), providing, for example,simultaneous isolation and pre-concentration of targeted proteins fromun-fractionated human plasma. Mass spectrometric detection allows, forexample, not only protein profiling but also screening of glycanstructure micro-heterogeneity of targeted antigens. As shown in theExamples, assays with mNPs achieved subnanomolar (10⁻⁹-10⁻¹⁰ M) limitsof detection and showed good extraction efficiency for plasma proteinthat was highly diluted (e.g. 500-fold). With this level of sensitivity,the two exemplary proteins in human plasma targeted in the Examplescould be analyzed unambiguously. This indicates that the mNPs+massspectrometry approach of the present invention can be used to rapidlyscreen relatively low-level targeted proteins in complex mixturescontaining other high-abundance proteins such as serum albumin. Comparedwith commercially available microscale particles, theantibody-conjugated mNPs of the present invention exhibitedsignificantly better extraction efficiency and specificity.

The advantages of the mNP plus mass spectrometry approach of the presentinvention are numerous. For example, manufacturing costs for each assay(e.g. screening a plasma sample) may be low. Another advantage is that auser may be able to complete an assay in less than about 20 minutes.Another advantage is the ability to use the assays of the presentinvention in a high-throughput nanoarray format. A further advantage ofmany embodiments of the present invention is the ability to use aslittle as 1 μL of plasma from healthy individuals or patients togenerate a target analyte profile.

The remainder of the description of the invention is provided below inthe following sections: I. Magnetic Nanoparticles; II. Target Analytesand Ligand Molecules; and III. Mass Spectrometry with Ligand ConjugatedMagnetic Nanoparticles.

I. Magnetic Nanoparticles

The magnetic nanoparticles of the present invention generally comprise asolid support magnetic core particle in the nanometer size range. Thecore particle employed to construct the ligand conjugated magneticnanoparticles of the present invention are preferably small particles(e.g. nanometer range) that effectively serve as a solid support orsolid phase for conjugation to a plurality of ligand molecules and usedin conjunction with mass spectrometric methods. Even though particlescan be of any size, the preferred size is 0.1-500 nanometers, preferably1-150 nanometers, more preferably 5-15 nanometers, and most preferablyabout 9.0 nanometers. The particles may be uniform (e.g., being aboutthe same size) or of variable size. Particles may be any shape (e.g.spherical or rod shaped), but are preferably made of regularly shapedmaterial (e.g. spherical). The particles of the present invention arepreferably composed of material that exhibits superparamagneticproperties.

Magnetic nanoparticles may be composed of any type of material thatexhibits magnetic properties. For example, the nanoparticles useful inthe present invention may be composed of a metal, such as iron, nickel,cobalt, and alloys of these metals. In certain embodiments, the magneticnanoparticles are composed of ceramic material. Preferably, the magneticnanoparticles are composed of material exhibiting superparamagneticproperties (e.g. particles that can be magnetized with an externalmagnetic field but dispersed simultaneously once the magnet is removed).

II. Target Analyte and Ligand Molecules

The present invention is not limited by the type of ligand moleculesconjugated to the magnetic nanoparticles, nor is the invention limitedby the type of target analyte that is detected by the mass spectrometricanalysis. The target analytes and ligand molecules, may be, for example,proteins (e.g. antibodies, antibody fragments or receptors),carbohydrates, lipids, or nucleic acids. Preferably, the target analyteor ligand molecule is a protein found in human blood plasma (see, e.g.,Anderson et al., Molecular & Cellular Proteomics, 3:311-326, 2004,herein incorporated by reference, including supplemental materialassociated with this reference). Examples of proteins in human plasmaare provided in Table 1.

TABLE 1 Accession Target Analyte or Ligand Molecules Number 60-kDa heatshock protein, mitochondrial precursor (Hsp60) (60-kDa chaperonin)P10809 (CPN60) (Heat shock protein 60) (HSP-60) (mitochondrial matrixprotein P1) (P60 lymphocyte protein) (hucha60) 70-kDa peroxisomalmembrance protein homolog (internal fragment) AAB27045 Actin,cytoplasmic 1 (β-actin) P02570 Adiponectin precursor (30-kDa adipocytecomplement-related protein) (ACRP30) Q15848 (adipose most abundant genetranscript 1) (apm-1) (gelatin-binding protein) Afamin precursor;α-albumin (Homo sapiens) NP_001124 α-1-acid glycoprotein 1 precursor(AGP 1) (orosomucoid 1) (OMD 1) P02763 α-1-antichymotrypsin precursor(ACT) P01011 α-1-antitrypsin precursor (α-1 protease inhibitor)(α-1-antiproteinase) P01009 (PRO0684/PRO2209) α-1B-glycoproteinprecursor (α-1-B glycoprotein) P04217 α-2-antiplasmin precursor(α-2-plasmin inhibitor) (α-2-PI) (α-2-AP) P08697 α-2-HS-glycoproteinprecursor (Fetuin-A) (α-2-Z-globulin) (Ba-α-2-glycoprotein) P02765(PRO2743) α-2-macroglobulin precursor (α-2-M) P01023 AMBP proteinprecursor [contains α-1-microglobulin (protein HC) (complex-formingP02760 glycoprotein heterogeneous in charge) (α-1 microglycoprotein);inter-α-trypsin inhibitor light chain (ITI-LC) (bikunin) (HI-30)]Angiotensinogen precursor [contains angiotensin I (Ang I); angiotensinII (Ang II); P01019 angiotensin III (Ang III) (Des-Asp[1]-angiotensinII)] Antithrombin-III precursor (ATIII) (PRO0309) P01008 ApolipoproteinA-I precursor (Apo-AI) P02647 Apolipoprotein A-II precursor (Apo-AII)(apoa-II) P02652 Apolipoprotein A-IV precursor (Apo-AIV) P06727Apolipoprotein B-100 precursor (Apo B-100) [contains: apolipoproteinB-48 (Apo B-48)] P04114 Apolipoprotein C-II precursor (Apo-CII) P02655Apolipoprotein C-III precursor (Apo-CIII) P02656 Apolipoprotein Dprecursor (Apo-D) (apod) P05090 Apolipoprotein E precursor (Apo-E)P02649 Apolipoprotein F precursor (Apo-F) Q13790 Apolipoprotein L1precursor (apolipoprotein L-I) (apolipoprotein L) (apol-I) (Apo-L)O14791 (apol) Apolipoprotein(a) precursor (EC 3.4.21.—) (Apo(a)) (Lp(a))P08519 ATP synthase β chain, mitochondrial precursor (EC 3.6.3.14)P06576 Atrial natriuretic factor precursor (ANF) (atrial natriureticpeptide) (ANP) P01160 (prepronatriodilatin) [contains:cardiodilatin-related peptide (CDP)] β-2-glycoprotein I precursor(apolipoprotein H) (Apo-H) (B2GPI) (β(2)GPI) (activated P02749 proteinC-binding protein) (APC inhibitor) β-2-microglobulin precursor P01884Bullous pemphigoid antigen, human (fragment) I39467 C4b-binding proteinαchain precursor (c4bp) (proline-rich protein) (PRP) P04003 C4b-bindingprotein β chain precursor P20851 Calgranulin A (Migration inhibitoryfactor-related protein 8) (MRP-8) (cystic fibrosis P05109 antigen)(CFAG) (P8) (leukocyte L1 complex light chain) (S100 calcium-bindingprotein A8) (calprotectin L1L subunit) Carbonic anhydrase I; carbonicdehydratase (Homo sapiens) NP_001729 Carboxypeptidase N 83-kDa chain(carboxypeptidase N regulatory subunit) (fragment) P22792Carboxypeptidase N catalytic chain precursor (EC 3.4.17.3) (argininecarboxypeptidase) P15169 (kinase 1) (serum carboxypeptidase N) (SCPN)(anaphylatoxin inactivator) (plasma carboxypeptidase B) Cathepsin Dprecursor (EC 3.4.23.5) P07339 Cathepsin L precursor (EC 3.4.22.15)(major excreted protein) (MEP) P07711 Cathepsin S precursor (EC3.4.22.27) P25774 CCAAT/enhancer binding protein β, interleukin6-dependent NP_005185 CD5 antigen-like precursor (SP-α) (CT-2)(igm-associated peptide) 043866 Centromere protein F (350/400 kd,mitosin); mitosin; centromere NP_005187 Ceruloplasmin precursor (EC1.16.3.1) (ferroxidase) P00450 Chaperonin containing TCP1, subunit 4(δ); chaperonin NP_006421 Chloride channel Ka; chloride channel, kidney,A; hclc-Ka (Homo sapiens) NP_004061 Cholinesterase precursor (EC3.1.1.8) (acylcholine acylhydrolase) (choline esterase II) P06276(butyrylcholine esterase) (pseudocholinesterase) Clusterin precursor(complement-associated protein SP-40, 40) (complement cytolysis P10909inhibitor) (CLI) (NA1 and NA2) (apolipoprotein J) (Apo-J) (TRPM-2)Coagulation factor IX precursor (EC 3.4.21.22) (Christmas factor) P00740Coagulation factor V precursor (activated protein C cofactor) P12259Coagulation factor VIII precursor (procoagulant component)(antihemophilic factor) P00451 (AHF) Coagulation factor X precursor (EC3.4.21.6) (Stuart factor) P00742 Coagulation factor XII precursor (EC3.4.21.38) (Hageman factor) (HAF) P00748 Coagulation factor XIII A chainprecursor (EC 2.3.2.13) (protein-glutamine γ- P00488 glutamyltransferaseA chain) (transglutaminase A chain) Coagulation factor XIII B chainprecursor (protein-glutamine γ-glutamyltransferase B P05160 chain)(transglutaminase B chain) (fibrin stabilizing factor B subunit)Collagen α1(IV) chain precursor P02462 Complement C1r componentprecursor P00736 Complement C1s component precursor (EC 3.4.21.42) (C1esterase) P09871 Complement C2 precursor (EC 3.4.21.43) (C3/C5convertase) P06681 Complement C3 precursor (contains C3a anaphylatoxin)P01024 Complement C4 precursor (contains C4A anaphylatoxin) P01028Complement C5 precursor (contains C5a anaphylatoxin) P01031 Complementcomponent C6 precursor P13671 Complement component C7 precursor P10643Complement component C8 αchain precursor P07357 Complement component C8β chain precursor P07358 Complement component C8 γchain precursor P07360Complement component C9 precursor P02748 Complement factor B precursor(EC 3.4.21.47) (C3/C5 convertase) (properdin factor B) P00751(glycine-rich β glycoprotein) (GBG) (PBF2) Complement factor H precursor(H factor 1) P08603 Complement factor H-related protein (clone H 36-1)precursor, human (fragment) A40455 Complement factor I precursor (EC3.4.21.45) (C3B/C4B inactivator) P05156 Complement-activating componentof Ra-reactive factor precursor (EC 3.4.21.—) (Ra- P48740 reactivefactor serine protease p100) (rarf) (mannan-binding lectin serineprotease 1) (mannose-binding protein associated serine protease)(MASP-1) Copper-transporting ATPase 1 (EC 3.6.3.4) (copper pump 1)(Menkes disease-associated Q04656 protein) Corticosteroid-bindingglobulin precursor (CBG) (transcortin) P08185 C-reactive proteinprecursor P02741 Creatine kinase, M chain (EC 2.7.3.2) (M-CK) P06732Cytosolic purine 5′-nucleotidase (EC 3.1.3.5) P49902 D60S N-terminallobe human lactoferrin 1DSN Dopamine β-monooxygenase precursor (EC1.14.17.1) (dopamine β-hydroxylase) (DBH) P09172 Endothelin convertingenzyme (EC 3.4.24.—) 1, umbilical vein endothelial cell form, JC2521human Extracellular matrix protein 1 isoform 1 precursor; secretoryNP_004416 Fibrinogen α/α-E chain precursor (contains fibrinopeptide A)P02671 Fibrinogen β chain precursor (contains fibrinopeptide B) P02675Fibronectin precursor (FN) (cold-insoluble globulin) (CIG) P02751Fibulin-1 precursor P23142 Ficolin 3 precursor (collagen/fibrinogendomain-containing protein 3) O75636 (collagen/fibrinogendomain-containing lectin 3 P35) (Hakata antigen) Follitropin β chainprecursor (follicle-stimulating hormone β subunit) (FSH-β) (FSH-B)P01225 Gamma enolase (EC 4.2.1.11) (2-phospho-D-glycerate hydrolyase)(neural enolase) P09104 (NSE) (enolase 2) Gelsolin precursor, plasma(actin-depolymerizing factor) (ADF) (Brevin) (AGEL) P06396 Glialfibrillary acidic protein, astrocyte (GFAP) P14136 Glutamatecarboxypeptidase II (EC 3.4.17.21) Q04609 Glutamyl aminopeptidase (EC3.4.11.7), human A47531 Glycosylphosphatidylinositol specificphospholipase D1 isoform 1 NP_001494 GP120, IHRP = ITI heavychain-related protein (internal fragment) AAB34872 Gravin, human JW0057Haptoglobin-1 precursor P00737 Hemoglobin αchain P01922 Hemoglobin βchain P02023 Hemopexin precursor (β-1B-glycoprotein) P02790 Heparincofactor II precursor (HC-II) (protease inhibitor leuserpin 2) (HLS2)P05546 Hepatocyte growth factor activator precursor (EC 3.4.21.—) (HGFactivator) (HGFA) Q04756 HGF activator like protein (hyaluronan bindingprotein 2) Q14520 Histidine-rich glycoprotein precursor(histidine-proline rich glycoprotein) (HPRG) P04196 Human psoriasin(s100a7) P31151 Hypothetical protein dkfzp564a2416.1, human (fragment)T14738 Hypothetical protein dkfzp586m121.1, human (fragment) T08772Hypothetical protein KIAA0437, human (fragment) T00063 Immunoglobulinκchain, human S40354 Immunoglobulin J chain P01591 Inhibin β A chainprecursor (activin β-A chain) (erythroid differentiation protein) (EDF)P08476 Insulin-like growth factor binding protein 3 precursor (IGFBP-3)(IBP-3) (IGF-binding P17936 protein 3) Insulin-like growth factorbinding protein 5 precursor (IGFBP-5) (IBP-5) (IGF-binding P24593protein 5) Insulin-like growth factor binding protein complex acidlabile chain precursor (ALS) P35858 Insulin-like growth factor IAprecursor (IGF-IA) (somatomedin C) P01343 Inter-α-trypsin inhibitorheavy chain H1 precursor (ITI heavy chain H1) (Inter-α-inhibitor P19827heavy chain 1) (Inter-α-trypsin inhibitor complex component III)(serum-derived hyaluronan-associated protein) (SHAP) Inter-α-trypsininhibitor heavy chain H2 precursor (ITI heavy chain H2)(inter-α-inhibitor P19823 heavy chain 2) (inter-α-trypsin inhibitorcomplex component II) (serum-derived hyaluronan-associated protein)(SHAP) Inter-α-trypsin inhibitor heavy chain H3 precursor (ITI heavychain H3) (Inter-α-inhibitor Q06033 heavy chain 3) (serum-derivedhyaluronan-associated protein) (SHAP) Inter-α-trypsin inhibitor heavychain H4 precursor Q14624 Interferon-induced viral resistance proteinmxa, human A33481 Interleukin-12 αchain precursor (IL-12A) (cytotoxiclymphocyte maturation factor 35- P29459 kDa subunit) (CLMF p35) (NK cellstimulatory factor chain 1) (NKSF1) Interleukin-15 precursor (IL-15)P40933 Interleukin-6 precursor (IL-6) (B-cell stimulatory factor 2)(BSF-2) (interferon β-2) P05231 (hybridoma growth factor) Keratin 10,type I, cytoskeletal (clone HK51), human (fragment) PC1102 Kinesinfamily member 3A; kinesin family protein 3A (Homo sapiens) NP_008985Kininogen precursor (α-2-thiol proteinase inhibitor) (containsbradykinin) P01042 Leucine-rich α-2-glycoprotein precursor (LRG) P02750Lipopolysaccharide-binding protein precursor (LBP) P18428 L-lactatedehydrogenase B chain (EC 1.1.1.27) (LDH-B) (LDH heart subunit) (LDH-H)P07195 Lumican precursor (keratan sulfate proteoglycan lumican) (KSPGlumican) P51884 Melanoma-associated antigen p97 isoform 1, precursorNP_005920 Microtubule-associated protein τ(neurofibrillary tangleprotein) (Paired helical filament-τ) P10636 (PHF-τ) Mismatch repairprotein MSH2, human I37550 Mitotic kinesin-like protein-1 (kinesin-likeprotein 5) Q02241 Monocyte differentiation antigen CD14 precursor(myeloid cell-specific leucine-rich P08571 glycoprotein) Myosin heavychain, nonmuscle type A (cellular myosin heavy chain, type A) (nonmuscleP35579 myosin heavy chain-A) (NMMHC-A) Myosin heavy chain, skeletalmuscle, adult 1 (myosin heavy chain iix/d) (myhc-iix/d) P12882 Oxygenregulated protein precursor; oxygen regulated protein NP_006380Parathyroid hormone precursor (Parathyrin) (PTH) (Parathormone) P01270Peroxiredoxin 3; antioxidant protein 1; thioredoxin-dependent NP_006784Peroxisome proliferator-activated receptor binding protein (PBP) (PPARbinding protein) Q15648 (thyroid hormone receptor-associated proteincomplex component TRAP220) (thyroid receptor interacting protein 2)(TRIP2) (p53 regulatory protein RB18A) Phosphatidylcholine-sterolacyltransferase precursor (EC 2.3.1.43) (lecithin-cholesterol P04180acyltransferase) (phospholipid-cholesterol acyltransferase)Phosphodiesterase 5A isoform 1; cgmp-binding cgmp-specific NP_001074Phosphoglycerate mutase 2 (EC 5.4.2.1) (EC 5.4.2.4) (EC 3.1.3.13)(phosphoglycerate P15259 mutase isozyme M) (PGAM-M) (BPG-dependent PGAM2) (muscle-specific phosphoglycerate mutase) Phosphoinositide-3-kinase,catalytic, αpolypeptide NP_006209 Pigment epithelium-derived factorprecursor (PEDF) (EPC-1) P36955 Plasma kallikrein precursor (EC3.4.21.34) (plasma prekallikrein) (kininogenin) (Fletcher P03952 factor)Plasma protease C1 inhibitor precursor (C1 Inh) (C1 Inh) P05155 Plasmaretinol-binding protein precursor (PRBP) (RBP) (PRO2222) P02753 Plasmaserine protease inhibitor precursor (PCI) (protein C inhibitor)(plasminogen P05154 activator inhibitor-3) (PAI3) (acrosomal serineprotease inhibitor) Plasminogen activator inhibitor-1 precursor (PAI-1)(endothelial plasminogen activator P05121 inhibitor) (PAI) Plasminogenprecursor (EC 3.4.21.7) (contains angiostatin) P00747 Platelet basicprotein precursor (PBP) (small inducible cytokine B7) (CXCL7) P02775Platelet factor 4 precursor (PF-4) (CXCL4) (oncostatin A) (Iroplact)P02776 Platelet-activating factor acetylhydrolase IB αsubunit (EC3.1.1.47) (PAF acetylhydrolase P43034 45 kDa subunit) (PAF-AH 45-kDasubunit) (PAF-AH α) (PAFAH α) (Lissencephaly-1 protein) (LIS-1)Platelet-derived growth factor receptor αprecursor (Homo sapiens)NP_006197 Plectin 1, intermediate filament binding protein 500 kDa;plectin 1 NP_000436 Pregnancy zone protein precursor P20742Prostate-specific antigen precursor (EC 3.4.21.77) (PSA)(γ-seminoprotein) (kallikrein 3) P07288 (semenogelase) (seminin) (P-30antigen) Protein disulfide isomerase precursor (PDI) (EC 5.3.4.1)(prolyl 4-hydroxylase β subunit) P07237 (cellular thyroid hormonebinding protein) (P55) Protein kinase, camp-dependent, regulatory, typeI, α NP_002725 Protein tyrosine phosphatase; ptpase (Homo sapiens)AAB22439 Prothrombin precursor (EC 3.4.21.5) (coagulation factor II)P00734 Putative serum amyloid A-3 protein P22614 Receptorprotein-tyrosine kinase erbb-2 precursor (EC 2.7.1.112) (p185erbb2) (NEUP04626 proto-oncogene) (C-erbb-2) (tyrosine kinase-type cell surfacereceptor HER2) (MLN 19) Rho-associated, coiled-coil containing proteinkinase 1; p160rock NP_005397 Selenoprotein P precursor (sep) P49908Serine (or cysteine) proteinase inhibitor, clade A (α-1) NP_006206Serotransferrin precursor (transferrin) (siderophilin) (β-1-metalbinding globulin) P02787 (PRO1400) Serum albumin precursor P02768 Serumamyloid A protein precursor (SAA) (contains amyloid protein A (amyloidfibril P02735 protein AA) Serum amyloid A-4 protein precursor(constitutively expressed serum amyloid A protein) P35542 (C-SAA) Serumamyloid P-component precursor (SAP) (9.5S α-1-glycoprotein) P02743 Serumparaoxonase/arylesterase 1 (EC 3.1.1.2) (EC 3.1.8.1) (PON 1) (serumP27169 aryldiakylphosphatase 1) (A-esterase 1) (aromatic esterase 1)(K-45) Sex hormone-binding globulin precursor (SHBG) (sexsteroid-binding protein) (SBP) P04278 (testis-specific androgen-bindingprotein) (ABP) Signal recognition particle receptor αsubunit (SR-α)(docking protein α) (DP-α) P08240 Similar to human hsgcn1 U77700 (PID:g2282576); similar to yeast AAC83183 SPARC precursor (secreted proteinacidic and rich in cysteine) (osteonectin) (ON) P09486 (basementmembrane protein BM-40) Squamous cell carcinoma antigen 1 (SCCA-1)(protein T4-A) P29508 SWI/SNF-related matrix-associated actin-dependentregulator of NP_003060 Tetranectin precursor (TN) (plasminogen-kringle 4binding protein) P05452 Thrombospondin 1 precursor P07996 Thyroglobulinprecursor P01266 Thyroxine-binding globulin precursor (T4-bindingglobulin) P05543 Transthyretin precursor (prealbumin) (TBPA) (TTR)(ATTR) P02766 Trypsin 1 precursor (EC 3.4.21.4) (cationic trypsinogen)P07477 Vascular cell adhesion protein 1 precursor (V-CAM 1) (CD106antigen) (INCAM-100) P19320 Vinculin (metavinculin) P18206 VitaminD-binding protein precursor (DBP) (group-specific component)(GC-globulin) P02774 (VDB) Vitamin K-dependent protein S precursorP07225 Vitamin-K dependent protein C precursor (EC 3.4.21.69)(autoprothrombin IIA) P04070 (anticoagulant protein C) (bloodcoagulation factor XIV) Vitronectin precursor (serum spreading factor)(S-protein) (V75) (contains vitronectin P04004 V65 subunit; vitronectinV10 subunit; somatomedin B) V-kit Hardy-Zuckerman 4 feline sarcoma viraloncogene homolog NP_000213 Von Willebrand factor precursor (vwf) P04275Zinc-α-2-glycoprotein precursor (Zn-α-2-glycoprotein) (Zn-α-2-GP) P25311

In preferred embodiments, the magnetic nanoparticles are conjugated toantibodies or antibody fragments (e.g. antibodies or antibody fragmentsdirected toward the molecules in Table 1). The antibodies and antibodyfragments may be, for example, both polyclonal and monoclonalantibodies. Polyclonal antibodies may be raised, for example, in animalsby multiple subcutaneous or intraperitoneal injections of the relevantantigen (e.g. proteins in Table 1) and an adjuvant. It may be useful toconjugate the relevant antigen to a protein that is immunogenic in thespecies to be immunized (e.g. keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor) using a bifunctionalor derivatizing agent. In some embodiments, monoclonal antibodies areconjugated to the magnetic nanoparticles of the present invention.Monoclonal antibodies may be made, for example, in a number of ways,including using the hybridoma method (e.g. as described by Kohler etal., Nature, 256: 495, 1975, herein incorporated by reference), or byrecombinant DNA methods (e.g., U.S. Pat. No. 4,816,567, hereinincorporated by reference).

In certain embodiments, the target analytes or ligand molecules areacute phase reactant molecules (or antibodies or antibody fragmentsthereto) found in plasma. The acute-phase response, the biosyntheticprofiles of particular plasma proteins, involves the nonspecificphysiological and biochemical responses of endothermic animals to mostforms of tissue damage, infection, inflammation, and malignantneoplasia. In particular, the synthesis of a number of proteins israpidly up-regulated, principally in hepatocytes, under the control ofcytokines originating at the site of pathology. These proteins aretermed acute-phase reactants (APRs) which have been divided intopositive APR (one whose plasma concentration increases) and negative APR(one whose plasma concentration decreases). In mammals, typical positiveAPR including serum amyloid A protein (SAA), C-reactive protein (CRP)and serum amyloid P component (SAP) increases in plasma concentration inthe magnitude between 50% to 1000-fold. The patterns of cytokineproduction and acute-phase response differ in different inflammatoryconditions. Accordingly, acute-phase changes, as detected by the methodsof the present invention, should reflect the presence and intensity ofinflammation and therefore can be used as a clinical guide to diagnosis.As such, in some embodiments, the methods of the present invention areused to provide an APR plasma profile of a patient (e.g. to aid in thediagnosis or treatment of the patient).

In particular embodiments, the methods and compositions of the presentinvention are directed toward detecting the presence of absence ofC-reactive protein (CRP). CRP belongs to the family of pentraxin whichis named for its capacity to precipitate the somatic C-polysaccharide ofStreptococcus pneumonia. The pentraxin family is highly conservedthroughout nature and is known for its calcium-dependent ligand bindingand lectin properties. CRP was one of the first acute-phase proteins tobe described and is an exquisitely sensitive systemic marker ofinflammation and tissue damage. CRP is the major APR in humans with meanconcentration in human plasma is 0.8 mg/L. Following an acute-phasestimulus, the CRP level may increase as much as 10,000 fold (see, Pepys,et al., J. Exp. Clin. Invest, 2003, 111:1805-1812, herein incorporatedby reference). CRP concentration is a useful non-specific biochemicalmarker of inflammation. As such, detecting CRP by the methods of thepresent invention allows, for example, one to (a) screen for organicdisease, (b) monitor the response of treatment of inflammation andinfection, and (c) detect intercurrent infection in diseasescharacterized by modest or absent acute-phase responses.

In certain embodiments, the methods and compositions of the presentinvention are directed toward detecting the presence of absence of serumamyloid P component (SAP). SAP is another member of the pentraxin familyand is named for its universal presence in amyloid deposits such asAlzheimer's disease and the transmissible spongiform encephalopathies,where it is bound to amyloid fibrils. SAP is a pentameric glycoproteinin human plasma. SAP is non-acute phase protein in human and otherspecies, but is the major APR in mice. SAP is highly resistant toproteolysis and its binding to amyloid fibrils is proposed to protect itfrom degradation and contribute to the presence of amyloid deposits,leading it to become the target of a new anti-Alzheimer's therapy (see,Pepys et al., Nature, 2002, 417:254-259, herein incorporated byreference).

III. Mass Spectrometry with Ligand Conjugated Magnetic Nanoparticles

The present invention provides methods of screening a sample for targetanalytes using ligand conjugated magnetic nanoparticles and massspectrometry. FIG. 9 provides an exemplary embodiment of the methods ofthe present invention. As shown in this figure, a core Fe₃O₄ magneticnanoparticle that is amino functionalized with a silicon coating isexposed to a linker molecule (DSS in this figure) to create a linkerfunctionalized magnetic nanoparticle. This particle is then reacted witha plurality of ligand molecules (antibodies in FIG. 9) to create aligand conjugated magnetic nanoparticle. Empty binding sites on thisparticle are then blocked with a blocking molecule (methoxy ethyleneglycol is shown as an example in FIG. 9). This ligand conjugatedmagnetic nanoparticle is then exposed to a sample that contains a targetanalyte (target antigen in FIG. 9). The magnetic nanoparticle, withbound target analyte, may be mixed with MALDI-TOF matrix material (e.g.SA) and then subjected to mass spectrometric analysis (e.g. MALDI-TOFanalysis is shown in FIG. 9). In this regard, the presence of the targetanalyte can be detected in the sample.

The present invention is not limited by the nature of the massspectrometry technique utilized for analysis of the ligand conjugatedmagnetic nanoparticles. For example, techniques that find use with thepresent invention include, but are not limited to, ion trap massspectrometry, ion trap/time-of-flight mass spectrometry, matrix assistedlaser desorption/ionization (MALDI), MALDI-TOF, MALDI-TOF-TOF,quadrupole and triple quadrupole mass spectrometry, Fourier Transform(ICR) mass spectrometry, and magnetic sector mass spectrometry. Thoseskilled in the art will appreciate the applicability of other massspectroscopic techniques to such methods.

In preferred embodiments, matrix assisted laser desorption/ionization(MALDI) type mass spectrometry is employed. MALDI mass spectrometry canbe divided into two steps. The first step involves preparing a sample bymixing the ligand conjugated magnetic nanoparticles of the presentinvention, which may be bound to an target analyte, with a molar excessof matrix material. The matrix material is generally an organic acidwith a strong absorption at the wavelength of the laser being used. Themixture is allowed to dry and the resultant nanoprobe-target complex isembedded in the matrix crystal. The second step generally involvesdesorption of bulk portions of the solid sample by intense pulses oflaser light. The irradiation by the laser (typically 3-5 ns) inducesrapid heating of the matrix crystals, resulting in localized sublimationof matrix/target protein (protein fragment) crystals, and entrainingintact analyte into the expanding matrix plume. In preferredembodiments, MALDI mass spectrometry is combined with time of flight(TOF) analysis.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); 1 or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); DS (dextran sulfate); and C (degrees Centigrade).

Example 1 Mass Spectrometry Detection with Magnetic NanoparticlesDisplaying Antibodies

This example describes construction of various antibody displayingmagnetic nanoparticles and their use to detect target analyte moleculesin samples using mass spectrometry. In particular, this exampledescribes the construction of magnetic nanoparticles conjugated withanti-SAP or anti-CRP antibodies and the use of these nanoparticles todetect SAP and CRP in biological samples using mass spectrometry.

Materials and Methods

Materials. Cytochrome c, myoglobin, enolase, and sinapinic acid (SA)were purchased from Sigma-Aldrich (Mississauga, ON, Canada). SAP and CRPwere obtained from Calbiochem (San Diego, Calif., USA). Polyclonalanti-SAP was purchased from DakoCytomation (Carpinteria, Calif., USA).Monoclonal anti-CRP was purchased from Biodesign (Kennebunk, Me., USA).The magnetic Separator was from Qiagen (Valencia, Calif., USA).

Synthesis of Antibody-Conjugated Magnetic Nanoparticles. Iron oxidenanoparticles (Fe₃O₄) were synthesized by co-precipitation using FeCl₂and FeCl₃ under basic conditions (see, Kang et al., Chem. Mater, 1996,8:2209-2211, herein incorporated by reference). During preparation, thedesired “core” mNPs were treated with tetraethyl orthosilicate (TEOS) tocreate the silica-coated surface. Subsequently,3-aminopropyltrimethoxysilane (APS) was added for aminosilanemodification to give mNPs.

The antibody of interest was covalently linked to the mNPs surfacethrough the cross-linker bis(N-hydroxysuccinimide ester) (NHS-ester;DSS; see FIG. 1A). In brief, aminosilane-modified mNPs (0.5 mg) weredissolved in DMSO (0.5 mL) and immediately incubated with DSS solutionfor 1 hour at room temperature. This solution was then dried andresuspended with either anti-SAP solution (8.1 mg/mL, 25 μL) or anti-CRPsolution (15.4 mg/mL, 15 μL). The mixture was incubated at 4° C. forovernight. The cross-linker is active for primary amines and thus canbridge between the aminosilane-modified mNPs and antibodies (or otherproteins). Finally, the antibody-conjugated mNPs products weremagnetically separated and extensively washed with PBS (0.1 M, pH 7.4)to remove excess reactants. The final product was dried and stored at 4°C. for further use.

To compare with magnetic microbeads, commercially available magneticmicrobeads (Dynal Biotech, 2.8 μm) were conjugated with antibody asdescribed above.

Immunoaffinity Capture of Antigens. For the protein pool experiments, 10μg mNPs were immersed in 60 μL of PBS (pH 7.4), containing 0.5 μMmyoglobin, 0.1 μM CRP, 0.67 μM SAP, and 2.1 μM enolase for 60 minutes atroom temperature. Unbound proteins were removed by isolating the mNPsusing a magnetic separator. The mNPs were then washed four times with100 μL of 25 mM ammonium bicarbonate. For subsequent MALDI MS analysis,the mNPs were directly mixed with 1˜2 μL MALDI matrix SA (10 mgsinapinic acid dissolved in 1 mL solution containing 50% acetonitrile,50% water, and 0.1% trifluoroacetic acid), spotted onto the sampleplate, air dried and analyzed. For human plasma analyses, aliquots (5-20μL) of plasma, diluted in PBS (pH 7.4), were mixed with mNPs (conjugatedwith either anti-SAP or anti-CRP) and subjected to the sameimmunoaffinity reaction and MALDI MS analysis.

To evaluate the effect of incubation time, 1 μL aliquots of 50 μL (40ng/μL SAP) samples from supernatant were immediately transferred to thesample plate at different times: 0, 3, 10, 30 and 60 min. Each samplewas then applied with 0.5 μL MALDI matrix and subsequently analyzed byMALDI MS.

Mass Spectrometry. All mass spectra were acquired by using a MALDI-TOFmass spectrometer Voyager DE-STR (PerSeptive Biosystems, USA) equippedwith a 337-nm nitrogen laser. The spectra were recorded in the linearmode using an accelerating voltage of 25 kV, a 90% grid voltage, 0.3%guide wire voltage, 650 ns delay time and a low-mass gate of 5000 Da.External mass calibration was usually applied based on a mixture of tworeference proteins [cytochrome c (M.W.=12361) and myoglobin(M.W.=16952)] covering the m/z range of 5 kDa to 80 kDa. A typical massspectrum was obtained by averaging 250 laser shots followed by noisereduction and Gaussian smoothing using Data-Explorer software (AppliedBiosystems, Foster City, Calif., USA).

Plasma Samples. Plasma samples from four patients with gastric cancerand four healthy controls were obtained with informed consent from theDepartment of General Surgery, Tri-Services General Hospital, Taipei,Taiwan. The procedure was approved by the Review Boards of Tri-ServiceGeneral Hospital, National Defense Medical Center. Plasma levels of CRPwere measured by the latex-particle-enhanced immunonephelometric assayusing a nephelometer (Dade Behring, Marburg, Germany) (Juan et al.,Proteomics, 2004, 4:2766-2775, herein incorporated by reference). SAS8.0 statistical software (SAS Institute GmbH, Heidelberg, Germany) wasused for statistical analysis. The Wilcoxon rank sum test was used tocompare CRP levels between healthy donors and patients with gastriccancer.

Preparation and Characterization of Nanoparticles

The synthesis of antibody-conjugated mNPs is illustrated in FIG. 1A. Thesuperparamagnetic nanoparticles (Fe₃O₄) were synthesized as describedabove. Nanoprobe stability and specific activity are importantparameters for the engineering of an optimal nanoparticle-antibodyimmunoassay. In order to develop more stable antibody-nanoparticlebioconjugates, the antibodies of interest (anti-SAP and anti-CRP) werecovalently conjugated to the aminosilane-modified mNPs through thebifunctionally amine-active cross-linker (DSS) (see FIG. 1A). Atransmission electron micrograph of the antibody-conjugated mNPs wasobtained. Small particle size can reduce steric hindrance and thusimprove specific activity. The size-distribution histogram of the mNPs(FIG. 1B) indicates that the diameter of iron oxide “core” ranges from5-15 nm, with an average diameter of ˜9.7 nm. In general, choosingparticles below about 15 nm in diameter ensures theirsuperparamagnetism, which allows stability and dispersion upon removalof the magnetic field (see, Tartaj et al, J. Phys. D. Appl. Phys, 2003,36, R182-197, herein incorporated by reference). Approximately 46 μg(320 pmol) of anti-SAP was immobilized on the surface of 1.0 mg mNPs, asdetermined using the BCA protein assay. Similarly, ˜9.8 μg (66 pmol) ofmonoclonal anti-CRP was immobilized. These antibody-nanoparticlebioconjugates could be stored in PBS, pH 7.4, at 4° C. for at least sixmonths without decomposition.

Protein Pool Experiments

Aliquots of functionalized mNPs were incubated with a biological mediumcontaining the targeted antigen. After the immunoaffinity interaction,the antigen-nanoparticle complexes were separated using a magnet, andnon-antigenic proteins and interfering impurities were subsequentlyremoved by a series of washes, abrogating the need for purification anddesalting. Finally, the nanoparticles were directly mixed with matrixfor MALDI MS analyses. To mimic a complex biological medium, a proteinpool was prepared in 60 μL PBS (0.01 M, pH 7.4) composed of antigenicproteins (SAP, 20% molar fraction and CRP, 3% molar fraction) and twoother “non-antigenic” proteins, myoglobin (Myo, 15%) and enolase (Eno,62%). The abundance of the antigenic proteins was purposely reduced totest the extraction efficiency of the targeted protein.

Prior to affinity extraction, as shown in FIG. 2A, the MALDI spectrum ofthe protein mixture shows the complexity of the mixture, in which thetargeted antigen, CRP, was not observed due to its low abundance (3%molar fraction) and the ion suppression effect (see, Krause et al.,Anal. Chem., 1999, 71:4160-4165, herein incorporated by reference).Suppression effects are commonly observed in MALDI MS due to thepresence of salts, buffer or other more abundant species in complexbiological media (see, Knochenmuss et al., Chem. Rev. 2003, 103,441-452, herein incorporated by reference). The suppression effect canresult in reduced signal intensity or even disappearance of the targetedanalyte.

After affinity extraction, the MALDI spectrum in FIG. 2B reveals thespecificity of nanoscale immunoassay, where CRP was selectivelyconcentrated and detected with an excellent signal-to-noise ratio of822. No background peak between m/z 5000-50000 was observed in controlexperiments before the addition of the protein mixture, showing nodetectable “chemical noise” arising from the antibody-conjugated mNPs.The absence of other abundant proteins in FIG. 2B excluded nonspecificbinding arising from electrostatic attraction or hydrogen bonding. Theuse of mNPs as a MALDI substrate overcomes the suppression effectbecause the particles are washed extensively to remove salt andabundant/non-antigenic proteins from the biological sample. The cleanmass spectrum demonstrates the advantages of nanoprobe-based affinityextraction in providing simultaneous protein isolation, enrichment, andsample desalting without the necessity of additional elution steps. Ingeneral, antibody-antigen interactions are strong, having dissociationconstants (Kd) ranging from 10⁻⁷ to 10⁻¹¹ M. Most antibody-antigencomplexes can be dissociated at extreme pH (i.e., pH<2 or pH>12). The pHof matrix solution (SA) is typically less than 2, and thus may serve toelute the antigen bound to the antibody-conjugated mNPs.

Mass spectrometric detection is also ideal for characterizingposttranslational modifications that cannot be predicted from genomicinformation. The MALDI spectrum in FIG. 2C shows a cluster of peakscorresponding to several SAP variants from the affinity extraction usinganti-SAP-conjugated mNPs. The expanded view shows that mass spectrum isdominated by the mass of 25464±4 Da, consistent with the theoreticalvalue of 25462 Da, as calculated from the known sequence. Accompanyingthe major peak were two peaks at 25174 Da and 24881 Da, corresponding tomass shifts of 290 Da and 583 Da, respectively. Within the experimentaluncertainty, the shifts can be attributed to the loss of one or twosialic acid residues (each residue M.W.=291). In the linear mode, wefound that the mass resolution did not deteriorate when theantigen-bound mNPs were deposited on the MALDI probe. Similarly, a massaccuracy of 0.02% could be routinely obtained by external calibration,comparable to the mass accuracy of conventional MALDI detection. Thus,the “direct” analysis of mNPs does not diminish the performance of theMALDI MS.

Kinetic Study of the Immunoreaction

The use of antibody-conjugated mNPs significantly reduces samplehandling time. In immunological assays, the incubation of antibody andantigen is often the rate-limiting step (e.g. 30 min to overnight forconventional ELISA) (see, e.g., Wang et al., J. Agric. Food Chem., 2004,52:7793-7797, herein incorporated by reference). To evaluate theefficiency, the effect of incubation time on antibody-antigenrecognition was investigated. After incubation of antibody-conjugatedmNPs with antigen solution, the amount of remaining antigen was measuredby MALDI MS. FIG. 3 shows that the peak intensities, corresponding tounbound antigen (SAP) in solution, decreased dramatically as a functionof incubation time over 10 min, at which time free SAP was barelydetectable (signal to noise ratio was <3). Significantly, maximumbinding of CRP was almost completed in even shorter incubation time (<3min).

This approach directly detected specific antibody-captured antigens byMALDI MS without using a secondary antibody or a reporter reaction.Unlike conventional immunoassays such as ELISA, for which the overallprocess usually requires at least 4 hours, our mNP-based immunoassay canbe shortened within 15-20 minutes. Thus, this rapid and sensitiveapproach is amenable to clinical applications such as high-throughput orpopulation screening.

Detection Sensitivity

Another advantage of the nanoprobe-based immunoassay is the ability topreconcentrate the antigen from diluted medium to a small volume ofmNPs. To demonstrate this concentration effect, a series of solutionswith different SAP concentration (160 nM to 8 nM) were prepared bydiluting an equal quantity (8 pmol) of SAP into different volumes. FIG.4 shows the MALDI mass spectra of extracted SAP after preconcentrationusing anti-SAP-conjugated mNPs. By contrast, the SAP peak was barelydiscernable (or not detected) when the diluted samples were analyzed byconventional MALDI, as shown in the inset of each panel.

The lower limit of detection, for this particular example, was exploredusing different amounts of SAP. After affinity extraction, FIG. 5 showsthe mass spectra of 60 μL of SAP solution after a series of dilutionsranging from 54 μg/mL to 16 ng/mL (1.9 μM to 0.6 nM). The SAP signalsdecrease progressively with decreasing concentration. Strong peakintensities were observed in all spectra except that for the 0.6 nMsolution, which had a signal-to-noise ratio of 3. Theoretically, thesensitivity of the current example depends on the MALDI MS detectionsensitivity and the efficiency of affinity extraction. Assuming fullrecovery of all the SAP present in the 60 μL of diluted solution, thedetection limit in this example is estimated to be 36 fmol, which iscomparable to the detection limit by direct deposition of SAP onto theMALDI probe (data not shown). It is noteworthy that SAP and CRP levelsin sera from healthy individuals were about 1.6 μM and 40 nM,respectively.

Detection of CRP and SAP from Human Plasma

It is well recognized that the human plasma proteomics holds the promiseof both a revolution in disease diagnosis and therapeutic aspects.However, human plasma is a very complex mixture of proteins having awide and dynamic range of abundance of more than 10¹². Indeed, 22proteins constitute about 99% of the protein content in plasma, with theremaining 1% comprising low-abundance proteins that are of greatinterest as potential biomarkers (see, e.g., Tirumalai et al., Mol. CellProteomics, 2003, 2:1096-1103, herein incorporated by reference). Thus,the magnetic nanoparticles of the present invention are preferably usedto detect low level proteins in human plasma.

To evaluate the specificity of the methods described in this example, 5μL of plasma from a healthy subject was incubated sequentially withanti-SAP- and with anti-CRP-conjugated mNPs for affinity extraction ofSAP and CRP, respectively. Prior to immunoaffinity extraction, noprotein profile could be obtained from the stock plasma sample due tothe interference of the salt and other plasma components. The plasmasample was therefore diluted 200-fold to reduce the salt concentrationand subjected it to analysis. The protein profile in FIG. 6A shows thecommonly observed abundant plasma proteins, including human serumalbumin (HSA), apolipoprotein A-I (ApoA-I), hemoglobin alpha chain(Hb-A), hemoglobin beta chain (Hb-B) and transthyretin (TTR). Afterimmunoaffinity extraction, SAP was detected with concomitant depletionof other proteins of higher concentration (FIG. 6B). Similarly, FIG. 6Cshows an apparent peak for CRP, even though the level of this protein is40-fold significantly lower than that of SAP in healthy individuals.Although the analysis showed minor peaks due to nonspecific binding ofother plasma protein, they did not interfere with the unambiguousidentification of CRP and SAP.

Comparison Between Nanoscale Particles and Microscale Particles

Recently, absorption of peptides/proteins onto microscale, reverse-phasemagnetic particles was used to preconcentrate a dilute, contaminatedsample for peptide mass mapping via MALDI analysis (see, Doucette etal., Anal. Chem., 2000, 72:3355-3362; Yaneva et al., Anal. Chem. 2003,75:6437-6448; and Villanueva et al., Anal. Chem., 2004, 76:1560-1570;all of which are herein incorporated by reference). Approaches thatconjugate antibody to microbeads have been reported to capture antigensof interest, yet these direct covalent conjugation procedures sufferfrom high background from non-specific binding and low signal-to-noiseratio (see, Peter, et al., Anal. Chem., 2001, 73:4012-4019, hereinincorporated by reference). Thus, we compared microscales particle andnanoscale particles with regard to extraction efficiency and detectionspecificity. Anti-SAP antibody was conjugated to commercially availableanimated magnetic microbeads (2.8 μm) by the same modification processused for aminosilane mNPs. The amounts of immobilized anti-SAPantibodies were determined to be 47 μg/mg and 26 μg/mg for mNPs andmicrobeads, respectively, using the BCA protein assay. Thus, to ensurethat equal amounts of antibody were used for antigen capture, we used a1:1.8 volume ratio of nanoparticle/microbeads in parallel immunoassays.

As shown in FIGS. 6D and 6E, the signal intensity and signal-to-noiseratio were dramatically reduced in the mass spectrum ofmicrobead-captured SAP and CRP compared with the nanoparticleexperiments (FIGS. 6B and 6C). These results indicate that mNPs affordbetter affinity extraction of the targeted protein, thereby improvingthe detection limit (see, Soukka et al., Clin. Chem., 2001,47:1269-1278, herein incorporated by reference). The fact that the samenumber of antibodies were immobilized on the surfaces of the nanoscaleand microscale particles suggests that the multivalent interactionbetween the targeted antigen and antibody-conjugated nanoparticlesyields superior sensitivity. Additionally, compared with the slightlyreduced resolution using microbeads (see Papac et al., Anal. Chem. 1994,66:2609-2613, herein incorporated by reference), both mass resolutionand profile were maintained using mNPs, without apparent peak broadeningand/or mass shift.

Concentration Effect for Plasma Protein Profiling

Affinity extraction of targeted antigens using antibody-conjugated mNPsnot only isolates but also preconcentrates low-level antigens onto thenanoprobe. To quantify this concentration effect, equal amounts ofplasma (1 μL from each subject) were diluted 50-, 500- and 1000-fold inPBS and analyzed using the immunoassays of the present invention. Theplasma SAP level of this subject had been rigorously determined to be45.4±3.2 mg/L (1.8 μM). FIG. 7 shows the MALDI mass spectra of SAPextracted from each diluted sample. Incubation of the diluted plasmasamples with the antibody-conjugated mNPs resulted in selectiveconcentration of SAP, as demonstrated by the similar mass spectraprofiles up to 500-fold dilution (FIGS. 7A, 7B and 7C). In the 1000-folddiluted sample (1.8 nM SAP), however, the captured antigen showedsignificantly lower intensity in the mass spectrum (FIG. 7D). It may bethat this decreased recovery was a consequence of incomplete recovery ofthe mNPs from the curved wall of the microcentrifuge tube during thewashing steps due to the large initial volume. These results demonstratethat 1 μL of plasma is sufficient to unambiguously identify an antigenof interest using immunoassays of the present invention. Despite thedecreased recovery at 1000-fold dilution, the detection sensitivity(estimated to be 1.8 nM) was comparable to the sensitivity using aprotein standard (SAP), demonstrating that the assay is refractory tothe presence of highly abundant non-antigenic proteins, salts andbuffers in plasma.

Analysis of Clinical Samples

The performance of mNP immunoassays was assessed using authenticclinical samples—plasma from 4 healthy individuals and 4 patients withgastric cancer (FIG. 8). CRP and SAP were detected in all the healthyindividuals, despite the fact that the levels of a few of them werebelow the detection limit of ELISA (<0.159 mg/L) (see, Juan et al.,Proteomics, 2004, 4:2766-2775, herein incorporated by reference). Themeasured intensities for CRP were considerably higher in the gastriccancer patient samples than in the healthy control samples. By contrast,the SAP levels in the patients were lower than those in the healthyindividuals. These observed differences in protein levels are consistentwith the differential protein profiles of gastric cancer patients asassessed by comparative proteomic approaches (see, e.g., Solakidi etal., Clin. Biochem. 2004, 37:56-60, herein incorporated by reference).It is noted that the ion intensity measured by the nanoparticle-basedassay using a 20 μL sample correlated with the concentration measured byELISA, indicating that mNP immunoassay would be useful for quantitativeprotein profiling.

Example 2 Mass Spectrometric Detection with Antibody Conjugated MagneticNanoparticles Blocked with Methoxy-Ethylene Glycol

This example describes construction of various antibody conjugatedmagnetic nanoparticles blocked with methoxy-ethylene glycol and theiruse to detect antigens in biological samples using mass spectrometry. Inparticular, this example describes the construction of magneticnanoparticles conjugated to anti-SAP or anti-CRP antibodies and the useof these nanoparticles to detect SAP and CRP in human plasma samplesusing mass spectrometry.

Construction of MEG Protected Antibody Encapsulated MagneticNanoparticles

The synthesis of antibody-conjugated ion oxide NP is illustrated in FIG.9. Aminated Fe₃O₄ nanoparticles (1 mg) was dispersed into 250 uLdimethyl sulfoxide (DMSO) and sonicated for 30 minutes. Aftersonicating, suberic acid bis N-hydroxysuccinimide ester (DSS) (10 mg,0.03 mmol) was added to the solution and stirred for one hour at roomtemperature. The resulting nanoparticles were washed with DMSO (100 uL)for 3 times to remove excess DSS. 50 uL of anti-SAP (6.7 mg/1 mL)antibody was added to the black solid and then shaked at 4° C. for 30minutes. Then, 50 uL of 80 mM compound 1 (MEG; methoxy ethylene glycol)was added to the mixture and followed by shaking for 12 hours at 4° C.After filtration, the nanoparticle was washed with PBS (100 uL, PH 7.4,0.1 M) for 5 times to give anti-SAP MNP as black powder. Anti-CRPencapsulated mNPs blocked with MEG were constructed in similar fashion.

Immunoaffinity Identification by MALDI-TOF

Immunoagglutination. Aliquots (2 mL, 5 mg/mL) of functionalized MNPswere added in 60 mL phosphate buffered saline (PBS, pH˜7.4) solutioncomposed of human plasma (5 mL). The solution was incubated at roomtemperature for 1 hour with slow rotation. After that, the mNPs wereagglutinated at the wall of eppendorf using a magnetic separator so thatthe supernatant was removed by pipette. During the washing steps, theMNPs were resuspended sequentially in 100 mL of Tris buffer (100 mM),TBS buffer (with 0.05% Tween 20), and 25 mM NaHCO₃(aq) and agglutinatedto remove supernatant. In the final step, the MNPs were thoroughly anddirectly transferred to a MALDI plate. Aliquots (1 mL) of matrixsolution (sinapinic acid, 10 mg/mL containing 50% acetonitrile, 50%water, and 0.1% trifluoroacetic acid) were applied and subsequentlyanalyzed by MALDI MS. Mass Spectrometry. MALDI-TOF mass spectra wereacquired by a mass spectrometer (Voyager-DE STR, PerSeptive Biosystems,USA) equipped with a 337 nm nitrogen laser source. Measurements weretaken in linear, positive ion mode at 25 kV acceleration voltage, 90%grid voltage, 0.3% guide wire voltage, 650 ns delayed ion extraction anda low mass gate of 5000 Da. The cytochrome c (M.W.=12361 Da), myoglobin(M.W.=16952 Da) were used as external standards for mass calibration. Atypical mass spectrum was obtained by average of 250 laser shotsfollowed by noise reduction and Gaussian smoothing using Data Explorersoftware (Applied Biosystems, Foster City, Calif., USA).

Results and Discussion

Anti-SAP mNP was tested for its ability to bind SAP in plasma of healthyhumans. SAP is a biomarker related to Alzheimer's disease and type 2diabetes, with a concentration of 0-40 mg/L in blood of healthy humans.Anti-SAP mNP was incubated with 5 mL plasma for the SAP concentration as40 mg/L. After immunoaffinity interaction, the SAP-nanoparticlecomplexes were separated by the magnet and the non-antigeniccontaminants were subsequently washed out with 100 mM Tris buffer, TBSbuffer (Tris buffered saline: 50 mM Tris-HCl, 0.85% NaCl, pH 7.5) and 25mM NaHCO₃(aq). Finally, the captured targeted antigen on nanoparticleswas directly analyzed by MALDI-TOF MS. The MALDI mass spectrum of humanplasma prior to immunoaffinity extraction is shown in FIG. 10A, andrevealed that the most abundant proteins were human serum albumin (HAS,66 KDa) and Apo protein C-I (Apo C-I, 6.6 KDa). The targeted SAP wasbarely detectable on mass analysis. However, after immunoaffinityextraction by anti-SAP MNP, the SAP peak alone was found in the massspectrum, as shown in FIG. 10B. Thus, this method successfullydemonstrated enrichment of the targeted protein and depletion ofcontaminants, including other abundant non-antigenic proteins, salts,and buffer.

Various extents of nonspecific binding with HSA and Apo C-I wereobserved in mass spectra, as shown in FIG. 10B, when anti-SAP MNP wasused. In bioassays, bovine serum albumin (BSA) is usually used as ablocking agent to prevent non-specific interactions. Thus, anti-SAP MNPwas further coated with BSA and then tested for specificity inimmunoaffinity extraction. However, good suppression of the HSA peak wasnot obtained (FIG. 1C). Therefore, the small methoxy-ethylene glycolwith terminal amino functionality (MEG, compound 1 in FIG. 9) wassynthesized and reacted with the terminal N-hydroxysuccinimide linkerson nanoparticles which had been incubated with anti-SAP antibody for 30minutes at 4° C. The new blocking reagent (MEG) exhibited markedlyimproved specificity and prominent depletion of non-specific binding(FIG. 10D). Only 6 mg of the MNPs was used to obtain the signal oftarget antigen with an infinitesimal amount of targeted protein (66pg/mL) without detectable nonspecific binding.

To optimize MEG conjugation on the MNP surface for depletion ofnon-specific interaction, a series of concentrations (from 10 mM to 100mM) of MEG were reacted with anti-SAP MNP. Results showed that depletionof non-specific binding of MEG-coated anti-SAP MNP with HSA wasMEG-concentration-dependent. MEG at 40 mM exhibited the highest ratio ofspecific to non-specific binding, as shown in FIG. 11. Althoughdifferent concentrations of MEG were used to react with activated esteron the mNP surface, the amounts of anti-SAP antibody on MNP were thesame in each experiment. These results suggested that the maximum amountof antibody that was assembled on mNP within about 30 minutes.

Next, C-reactive protein (CRP) was tested in order to demonstrate thegeneral applicability of this method. CRP is an exquisitely sensitivesystemic marker of acute-phase inflammatory response, tissue injury, andcoronary artery disease (see, e.g., Berton et al., Am. Heart. J., 2003,145:1094-1101, herein incorporated by reference). In recent studies, ithas been shown that CRP concentration rises from less than 0.05 mg/L tomore than 500 mg/L in serum (Pepys et al., J. Clin. Invest., 2003, 111,1805-1812, herein incorporated by reference) with an acute-phasestimulus, and the level of CRP has also been found to correlate with theseverity, extent, and progression of many different diseases. With theneed for quick response and assay specificity, we first preparedanti-CRP-mNP for detecting CRP present in low abundance in a cancerpatient's serum (5 mL plasma sample was used), which featured a CRPconcentration of 40 mg/L by ELISA. Following extraction with anti-CRPMNP, though CRP was detected as the most prominent peak by this non-MEGprotected MNP, nonspecific binding with Apo C-I was still observed (FIG.12A). Following conjugation with 30 mM of MEG, however, theMEG-protected anti-CRP-MNP successfully decreased the nonspecificbinding peak and enhanced the CRP peak (FIG. 12B). Surprisingly,MEG-protected anti-CRP-MNP could detect low CRP concentration present inminute amounts (<0.9 mg/L) in healthy individuals (5 uL plasma samplewas used), though this is difficult to achieve with the ELISA assay usedin clinical setting. This is shown in FIG. 12C (without MEG blocking)and FIG. 12D (with MEG blocking).

Example 3 Multiplexed Mass Spectrometric Detection with AntibodyConjugated Magnetic Nanoparticles

This Example describes the multiplex detection of three different targetanalytes simultaneously using mass spectrometry and MEG-protectedantibody conjugated nanoparticles. After affinity extraction with amixture of anti-SAA, anti-CRP, and anti-SAP MNPs, SAA, CRP, and SAP wereselectively and simultaneously detected in 1 mL plasma (100-folddilution) obtained from a healthy individual, as shown in FIG. 13. Theasterisk represents the impurity peak generated from commerciallyavailable anti-SAP antibody. The heterogeneous profile of native SAP anddeglycosylated SAP variants (from removal of the terminal sialic acidresidue) was clearly resolved. Meanwhile, the truncated forms of SAAisotypes were present in the plasma from a healthy individual. Theresults of this example demonstrate the ability of the magneticnanoparticles of the present invention to be detected by massspectrometry in a multiplex manner.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in the artare intended to be within the scope of the following claims.

1. A method of assaying a target analyte in a sample comprising thesteps of: a) providing a composition comprising antibody-conjugatedmagnetic nanoparticles (Ab-mNPs), wherein each of the Ab-mNPs comprises:i) a core iron oxide nanoparticle ranging in size from 0.1 to 500 nm;ii) cross-linkers, covalently conjugated to the surface of the corenanoparticle; iii) antibody molecules specific for a target analyte,covalently conjugated to the cross-linkers; and iv) methoxy ethyleneglycol having the structure H₃C—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—NH₂covalently conjugated through the amine group to cross-linkers that areunconjugated to the antibody molecules; b) exposing a sample that maycontain the target analyte to the Ab-mNPs to allow the target analyte tobe captured by the Ab-mNPs; c) subjecting the Ab-mNPs and the samplefrom step (b) to a magnetic field to separate the Ab-mNPs and thecaptured target analyte from the rest of the sample; and d) performingmass spectrometry on the Ab-mNPs and the captured target analyte fromstep (c) to obtain a mass spectrum for assaying the target analyte;wherein the target analyte is a compound selected from the groupconsisting of a protein, a polypeptide and a peptide.
 2. The method ofclaim 1, wherein the sample is obtained from a patient suspected ofhaving, a change in the plasma level of the target analyte.
 3. Themethod of claim 2, wherein the target analyte is a compound selectedfrom the group consisting of serum amyloid P component (SAP) andC-reactive protein (CRP).
 4. The method of claim 2, further comprisingthe step of comparing the mass spectrum of the target analyte of thesample obtained from the patient with that obtained from a healthysubject to detect whether there is a change in the profile of the targetanalyte in the patient.
 5. The method of 1, wherein the providing stepfurther comprises the step of synthesizing the Ab-mNPs, which comprises:reacting the methoxy ethylene glycol with magnetic nanoparticles toobtain the Ab-mNPs with the methoxy ethylene glycol covalentlyconjugated through the amine group to the cross-linkers that areunconjugated to the antibody molecules wherein each of the magneticnanoparticles in the reacting step comprises: i) a core iron oxidenanoparticle; ii) cross-linkers, conjugated to the surface of the corenanoparticles; and iii) antibody molecules specific for the targetanalyte, covalently conjugated to the cross-linkers.
 6. The method ofclaim 5, wherein the concentration of the methoxy ethylene glycol forreacting with the magnetic nanoparticles is between 30 and 50 mM.
 7. Themethod of claim 1, wherein the sample is from a cancer patient.
 8. Themethod of claim 1, wherein the target analyte is a protein selected fromthe group consisting of serum amyloid P component (SAP), C-reactiveprotein (CRP), serum amyloid A protein (SAA), myoglobin, enolas (Eno),and apolipoprotein.
 9. The method of claim 1, wherein the target analyteis a human plasma protein.
 10. The method of claim 2, wherein the sampleis obtained from a cancer patient.
 11. A method for assaying multiplextarget analytes in a sample comprising the steps of: a) providing acomposition comprising more than one kind of Ab-mNPs, of which each kindhas a different antibody conjugated to it, and each of the Ab-mNPscomprises: i) a core iron oxide nanoparticle ranging in size from 0.1 to500 nm; ii) cross-linkers, conjugated to the surface of the corenanoparticle; iii) antibody molecules specific for one of the multiplextarget analytes, covalently bound to the cross-linkers; and iv) methoxyethylene glycol having the structure H₃C—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—NH₂covalently conjugated through amine group to the cross-linkers that areunconjugated to the antibody molecules; b) exposing a sample that maycontain the multiplex analytes to the Ab-mNPs to allow the multiplexanalytes to be captured by the Ab-mNPs; c) subjecting the Ab-mNPs andthe sample from step (b) to a magnetic field to separate the Ab-mNPs andthe captured multiplex analytes from the rest of the sample; and d)performing mass spectrometry on the Ab-mNPs and the captured multiplextarget analytes from step (c) to obtain mass spectra for assaying themultiplex target analytes; wherein the target analytes are compoundsselected from the group consisting of proteins, polypeptides and apeptides.
 12. The method of claim 11, wherein the sample is from apatient suspected of having a change in the plasma level of the targetanalytes.
 13. The method of claim 12, wherein the target analytes arehuman plasma proteins.
 14. The method of claim 13, further comprisingthe step of comparing the mass spectra of the target analytes of thesample obtained from the patient with those obtained from a healthysubject to detect whether there is a change in the profile of the targetanalytes in the patient.
 15. The method of claim 11, wherein the sampleis obtained from a cancer patient.